Abstract

Objectives We investigated the effect of reducing mitochondrial oxidative stress by the mitochondrial-targeted antioxidant peptide SS-31 in hypertensive cardiomyopathy.

Background Oxidative stress has been implicated in hypertensive cardiovascular diseases. Mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase have been proposed as primary sites of reactive oxygen species (ROS) generation.

Methods The mitochondrial targeted antioxidant peptide SS-31 was used to determine the role of mitochondrial oxidative stress in angiotensin II (Ang)-induced cardiomyopathy as well as in Gαq overexpressing mice with heart failure.

Results Ang induces mitochondrial ROS in neonatal cardiomyocytes, which is prevented by SS-31, but not the nontargeted antioxidant N-acetyl cysteine (NAC). Continuous administration of Ang for 4 weeks in mice significantly increased both systolic and diastolic blood pressure, and this was not affected by SS-31 treatment. Ang was associated with up-regulation of NADPH oxidase 4 (NOX4) expression and increased cardiac mitochondrial protein oxidative damage, and induced the signaling for mitochondrial biogenesis. Reducing mitochondrial ROS by SS-31 substantially attenuated Ang-induced NOX4 up-regulation, mitochondrial oxidative damage, up-regulation of mitochondrial biogenesis, and phosphorylation of p38 mitogen-activated protein kinase and prevented apoptosis, concomitant with amelioration of Ang-induced cardiac hypertrophy, diastolic dysfunction, and fibrosis, despite the absence of blood pressure-lowering effect. The NAC did not show any beneficial effect. The SS-31 administration for 4 weeks also partially rescued the heart failure phenotype of Gαq overexpressing mice.

Hypertension is a major global health issue, accounting for approximately one-half of the cases of stroke and ischemic heart disease and approximately 13% of total death worldwide (1). Systemic hypertension induces left ventricular hypertrophy (LVH), fibrosis, and diastolic dysfunction and increases the risk of coronary artery disease, which leads to congestive heart failure (2). The renin-angiotensin aldosterone system is the central regulator of hypertensive cardiovascular diseases. Angiotensin II (Ang) induces LVH, cardiac fibrosis, and diastolic dysfunction. At the molecular level, Ang binds to the angiotensin receptor-1 (ATR1), a guanine nucleotide-binding protein type q alpha subunit (Gαq)-coupled receptor, then stimulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases to produce reactive oxygen species (ROS) (3). Recent studies have reported that the nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) isoform of NADPH oxidase is present in mitochondria. The ROS generated by NADPH oxidase was shown to stimulate mitochondrial ROS production and induce mitochondrial dysfunction (4–7). Furthermore, we previously demonstrated that mitochondrial ROS plays a key role in aging and lifespan regulation, as shown by approximately 20% extension of lifespan in mice overexpressing catalase targeted to mitochondria (mCAT) (8). We further showed that mCAT attenuated age-dependent LVH and diastolic dysfunction, concomitant with attenuation of age-dependent increases in cardiac mitochondrial oxidative damage, without any effect on the increase of cardiac Ang observed in aged mice (9). Therefore, we hypothesized that Ang might induce mitochondrial ROS in cardiomyocytes and that scavenging mitochondrial ROS by a mitochondrial-targeted antioxidant peptide might be beneficial in the setting of hypertensive cardiomyopathy.

The Szeto-Schiller (SS)-31 peptide (D-Arg-2′,6′-dimethyl-tyrosine-Lys-Phe-NH2) belongs to a family of aromatic cationic peptides that selectively target to mitochondrial inner membrane and can scavenge superoxide, hydrogen peroxide, peroxynitrite, and hydroxyl radicals (10,11). The SS-31 has been shown to reduce mitochondrial ROS in epithelial, endothelial, and neuronal cells exposed to electron-transport-chain inhibitors as well as pro-oxidants including t-butyl-hydroperoxide and hypochlorous acid (12,13). The SS-31 also inhibits apoptosis mediated by mitochondrial release of cytochrome c elicited by mitochondrial permeability transition (11). In vivo administration of SS-31 has demonstrated efficacy in several animal models associated with mitochondrial oxidative stress, including reduction of ischemia-reperfusion injury (14), protection against neurodegeneration (15), and prevention of insulin resistance induced by high-fat diet (16). In this study, we demonstrated that Ang induced mitochondrial ROS in cardiomyocytes and increased cardiac mitochondrial protein oxidative damage. Reduction of mitochondrial ROS by SS-31 ameliorated Ang-induced cardiac hypertrophy, fibrosis, and apoptosis, concomitant with reduced activation of p38 mitogen-activated protein kinase (MAPK). The SS-31 administration also partially rescued the heart failure phenotype of mice with Gαq overexpression as a model of chronic catecholamine/Ang stimulation (17).

The FVBxC57BL/6J F1 hybrid mice (6 to 10/group, approximately 12 weeks of age) and wild-type littermates were treated with saline, Ang (1.1 mg/kg/day), Ang + SS-31 (3 mg/kg/day), Ang + NAC (approximately 500 mg/kg/day in drinking water) (18), Gαq (FVBxC57BL/6 F2), or Gαq +SS-31 (3 mg/kg/day). Ang and SS-31 were continuously administered for 4 weeks with subcutaneous Alzet 1004 osmotic minipumps (Alzet, Cupertino, California). Echocardiography was performed at baseline and 4 weeks after pump. As a reference for SS-31 effects, we included a genetic mouse model of Rosa-26 inducible-mCAT (C57BL/6), in which mitochondrial catalase was expressed 2 weeks before Ang treatment. Blood pressure (BP) was measured in a separate group of mice by telemetry with an intravascular catheter PA-C10 (DSI, St. Paul, Minnesota). After 4 weeks of treatment, mouse ventricles were harvested. Quantitative image analysis of trichrome staining was performed to evaluate the severity of fibrosis. Cardiac mitochondrial protein carbonyl content was analyzed with OxiSelect Protein Carbonyl ELISA Kit (Cell Biolabs, San Diego, California). Quantitative polymerase chain reaction (qPCR) was performed with Applied Biosystems 7900 thermocycler with Taqman Gene Expression Assays on Demand (Applied Biosystems, Foster City, California). Western blotting was used to analyze NOX4, cleaved caspase-3, phosphorylated, and total p38 MAPK.

Statistical analysis

All data are presented as mean ± SEM. Comparisons between 2 groups are performed with Student t tests. One-way analysis of variance was used to compare differences among multiple groups, followed by Tukey post hoc test for significance. All p values <0.05 were considered significant.

Results

Ang increased mitochondrial and total cellular ROS in neonatal cardiomyocytes, which was alleviated by SS-31

Flow cytometric analysis demonstrated that Ang increased MitoSOX fluorescence (an indicator of mitochondrial superoxide) (Fig. 1A) and DCFDA fluorescence (an indicator of total cellular ROS) (Fig. 1B) in neonatal cardiomyocytes by approximately 60% and 45%, respectively (p < 0.01 for both) (Fig. 1C). Treatment with NAC, a nontargeted antioxidant drug, did not show any significant effect on mitochondrial or total cellular ROS after Ang. In contrast, SS-31 significantly reduced Ang-induced MitoSOX and DCFDA fluorescence to near control levels (Fig. 1). Ang-induced MitoSOX and DCFDA fluorescence could also be attenuated by 4-Cl-benzodiazepam, an inhibitor of the inner mitochondrial anion channel, or cyclosporine A, an inhibitor of the mitochondrial permeability transition pore or diazoxide, an activator of the mitochondrial K-ATP channel (Online Figs. 1A to 1D and 1G)—further supporting mitochondrial mechanisms of ROS induction (see Discussion). In contrast, 5-hydroxydecanoate, an inhibitor of the mitochondrial K-ATP channel, did not significantly affect Ang-induced ROS (Online Figs. 1E and 1F).

After 4 weeks of Ang, echocardiography revealed an approximately 2-fold increase in left ventricular (LV) mass index compared with baseline (Fig. 3A), no change in LV end diastolic diameter (data not shown) or systolic function as measured by fractional shortening (FS) (Fig. 3B), and an approximately 35% decline in Ea/Aa, an indicator of diastolic function (Fig. 3C). Simultaneous administration of SS-31 significantly ameliorated Ang-induced cardiac hypertrophy and diastolic dysfunction, with a 33% reduction of LV mass index (Ang: 6.32 ± 0.39 vs. Ang + SS-31: 4.21 ± 0.17 mg/g, p = 0.001) (Fig. 3A, left panel) and better preservation of Ea/Aa (Ang: 0.723 ± 0.15 vs. Ang+SS-31: 1.17 ± 0.11, p = 0.04) (Fig. 3C, left panel). These effects were comparable to those of catalase targeted to mitochondria (inducible mCAT), in which induction of mitochondrial catalase 2 weeks before Ang also conferred protection against Ang-induced cardiac hypertrophy and diastolic dysfunction (Figs. 3A to 3D, right panels). To investigate whether the protective effect is specific to a mitochondrial targeted antioxidant, we administered the nontargeted antioxidant NAC in drinking water for 4 weeks simultaneously with the Ang pump. This treatment has been shown to ameliorate cardiomyopathy after acute aortic banding in mice (18); however, it did not show any beneficial effect on Ang-induced cardiomyopathy (Figs. 3A to 3C).

(A) Angiotensin II (Ang) for 4 weeks substantially increased left ventricular (LV) mass index in control mice. Simultaneous administration of SS-31 significantly attenuated this increase in LV mass index (left). This was to an extent similar to that observed in mice with inducible overexpression of mitochondrial catalase (i-mCAT) (right). (B) Fractional shortening (FS) was not significantly changed after 4 weeks of Ang in the presence or absence of mitochondrial antioxidants. (C) Diastolic function measured by tissue Doppler imaging of Ea/Aa was significantly reduced after Ang but was significantly ameliorated by SS-31 or genetic overexpression of mCAT. Administration of N-acetyl cysteine (NAC) for 4 weeks did not confer any significant protection for Ang-induced hypertrophy and diastolic dysfunction (light blue bar on left panel, A to C), n = 6 to 7.

Ang-Induced Cardiac Hypertrophy and Fibrosis Were Attenuated by SS-31 But Not Oral NAC

(A) Angiotensin II (Ang) significantly increased heart weight (normalized to tibia length), and this was significantly attenuated by SS-31 but not by N-acetyl cysteine (NAC). (B) Quantitative polymerase chain reaction (qPCR) showed a dramatic increase in atrial natriuretic peptide gene expression, which was significantly prevented by SS-31 but not by NAC. (C) Representative histopathology showing that Ang-induced substantial perivascular fibrosis (PVF) and interstitial fibrosis (IF), which was protected by SS-31 but not NAC. (D) Analysis of blue trichrome staining demonstrated a significant increase in ventricular fibrosis after Ang, and this was substantially attenuated by SS-31 but not NAC. (E) The qPCR showed up-regulation of pro-collagen1a2 messenger ribonucleic acid (mRNA) after Ang, which was significantly reduced in SS-31 hearts but not in NAC hearts; n = 4 to 7.

It has been proposed that Ang mediates its effects via Gαq. However, Gαq transgenic overexpression involves a persistent lifelong stimulation of Gαq, starting from developmental stage, and is likely a stronger stimulus than obtained with Ang. Thus, transgenic overexpression of Gαq displayed a much stronger phenotype than 4-week Ang, including systolic heart failure in mice by 14 to 16 weeks of age (17). The SS-31 for 4 weeks partially rescued the heart failure phenotype in mice overexpressing Gαq, including a significant amelioration of systolic dysfunction, cardiac hypertrophy, and myocardial performance (Figs. 7A, 7D, and 7E).

Central to the scheme hypothesized in Figure 8 is the amplification of mitochondrial ROS, which can occur by multiple mechanisms. Ang is a key mediator of hypertension that binds to ATR1, a Gαq coupled-receptor, then activates NADPH oxidase through a protein kinase C-dependent mechanism. Although the NOX2 isoform of NADPH oxidase has been shown to mediate Ang effects, the NOX4 isoform has recently been reported to increase in response to various hypertrophic stimuli (6) and is localized in mitochondria (7), and activation of this isoform increases mitochondrial ROS (6). Although our hypothesis indicates that ROS produced directly from NOX4 and/or indirectly by NOX2 isoform is amplified within mitochondria, the relative contribution of these isoforms in elevating ROS in mitochondria is not yet clear, and it is possible that NOX-independent mechanism(s) might also contribute to the Ang and Gαq phenotypes.

Because escape of electrons from the mitochondrial electron transport complexes is a principal source of cellular ROS, oxidative damage to these complexes has been proposed to lead to a vicious cycle (27). Another potential mechanisms of Ang-induced mitochondrial ROS could be the previously reported ROS-induced ROS release from mitochondria, which might be mediated by activation of the inner mitochondrial anion channel (inhibited by 4-chlorodiazepam) (28), or the mitochondrial permeability transition pore (inhibited by cyclosporine) (29), or mitochondrial KATP channels (activated by diazoxide) (4). The involvement of ROS-induced ROS release from mitochondria should be considered as a primary mechanism of Ang-induced ROS signaling (30), as confirmed by observations that Ang-induced mitochondrial-ROS was reduced by simultaneous treatment with diazoxide, 4-chlorodiazepam, or cyclosporine A (Online Fig. 1). This places mitochondria in a central position for signal amplification and, conversely, for therapeutic targeting. Although most antihypertensive medications act upstream at the receptor level (beta-blockers, calcium channel blockers, angiotensin receptor blockers) or even at remote sites of action (diuretics, angiotensin-converting enzyme inhibitor), antioxidants provide an alternate intervention strategy in cardiac hypertrophy and failure. Several antioxidant studies have demonstrated some protective effect in cardiovascular diseases. For instance, Euk-8, a superoxide dismutase and catalase mimetic, ameliorated heart failure in ROS-sensitive mouse models subjected to pressure overload (31). Zhou et al. (32) reported that overexpression of metallothionein suppressed oxidative and nitrosative stress, apoptosis, and pathological remodeling in response to a short-term sub-pressor dose of Ang. Our study, however, points to the rationale of targeting antioxidants to mitochondria. The mitochondrial antioxidant MitoQ has been shown to reduce BP in the spontaneous hypertensive rat, concomitant with improvement of endothelial function and reduction of heart weight; however, the mechanism of cardioprotection was unclear (33). Our study found no effect of SS-31 on BP but showed evidence of direct cardioprotective mechanisms involving reduction of cardiac mitochondrial damage, amelioration of apoptosis and fibrosis, and attenuation of p38 MAP kinase activation.

Hypertension is a highly prevalent disease that imposes a major risk for the development of atherosclerosis, cardiomyopathy, stroke, sudden cardiac death, and heart failure. Hypertension-induced heart failure might be manifested as systolic heart failure or heart failure with preserved ejection fraction (HFpEF); the latter accounts for nearly one-half of the patients with heart failure, and the prognosis of HFpEF is only marginally better than that of systolic heart failure (34). Although antihypertensive treatment reduces mortality and improves quality of life in patients with established systolic heart failure (35), there is no convincing evidence for any effective treatment of HFpEF. These underscore the urgent need to develop new prevention and treatment strategies for hypertensive cardiovascular diseases. Although clinical trials applying antioxidants to disease prevention in general and cardioprotection in particular (36) have been disappointing, these studies have generally used nontargeted cellular antioxidants. Our study demonstrates that a mitochondrial-targeted antioxidant drug is beneficial in preventing hypertension-induced target organ damage and provides a strong rationale for investigating its clinical application for the treatment or prevention of hypertensive cardiovascular diseases.

Appendix

For supplementary figures and Methods, please see the online version of this article.

Online Appendix

Footnotes

This work was supported by the National Institutes of Health Grants R01 HL101186, P30 AG013280, and P01 AG001751. Dr. Szeto is the inventor of SS-31, the Cornell Research Foundation (CRF) holds several patents covering the SS peptides, and a patent application has been filed for the findings described in this paper, with Drs. Szeto and Rabinovitch as inventors. The CRF has licensed the SS peptide technology for further research and development to a commercial enterprise in which CRF and Dr. Szeto have financial interests. All other authors have reported that they have no relationships to disclose.

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